There has been a frightening rise in the incidence of disease-causing bacteria resistant to commonly used antibiotics. We therefore urgently need to identify additional molecules to use as antibiotics. Bacteria themselves have historically been the best source for antibiotics and other related 'secondary metabolites'. While the most common and abundant microbial antibiotics have already been harvested as low-hanging fruit, microbes remain a deep potential source of new secondary metabolites. A critical barrier to obtaining these compounds, however, has been that bacteria rarely produce their full repertoire of secondary metabolites when cultured in the laboratory. What we lack therefore are efficient mechanisms to stimulate the synthesis of these dormant genes required to synthesize these additional, potentially novel, antibiotics. Bacteria grown in co-culture appear to up-regulate their production of secondary metabolites to act as interspecies signaling cues. Our central hypothesis is that co-culture will stimulate the production of molecules not produced in mono-culture, some of which may be previously uncharacterized antibiotics. We will test this hypothesis using the following approaches. In Aim 1 we will use imaging mass spectrometry to broadly detect the production of compounds produced specifically in co-culture, while in Aim 2 we will take a targeted approach and use bioinformatics to identify bacterial strains encoding proteins predicted to create novel secondary metabolites, and use co-culture to stimulate their production. Finally, in Aim 3 we will test the various co-culture-induced metabolites obtained in the previous two aims for antibiotic activity against human pathogens. The most promising leads will be isolated and chemically identified. Our research proposes to exploit microbial co-culture to elicit the production of bacterial metabolites to identify newly discovered antibiotics. Ultimately, the results from this research will contribute to our long-term goal of ensuring that microbes continue to provide us with the building blocks necessary to replenish our arsenal of antibiotics for therapeutic use.